Rhodococcus equi is a multi-host pathogen that infects a range of animals as well as immune-compromised humans. Equine and porcine isolates harbour a virulence plasmid encoding a homologous family of virulence-associated proteins associated with the capacity of R. equi to divert the normal processes of endosomal maturation, enabling bacterial survival and proliferation in alveolar macrophages. To provide a basis for probing the function of the Vap proteins in virulence, the crystal structure of VapD was determined. VapD is a monomer as revealed by multi-angle laser light scattering. The structure consists of an elliptical, compact eight-stranded β-barrel with a novel strand topology and pseudo-twofold symmetry, suggesting evolution from an ancestral dimer. Surface-associated octyl-β-D-glucoside molecules may provide clues to function. Circular-dichroism spectroscopic analysis indicates that the β-barrel structure is preceded by a natively disordered region at the N-terminus of the protein. Sequence comparisons show that the core folds of the other plasmid-encoded virulence-associated proteins from R. equi strains are similar to that VapD, and that sequences encoding putative R. equi Vap-like proteins occur in other, diverse bacterial species. The functional implications of the structure will be discussed in the light of the unique structural features of VapD and its partial structural similarity to other β-barrel proteins.